Multiplex competition assay for profiling binding epitopes of affinity agents for clinical diagnostics use

Abstract

Some embodiments of the systems and methods provided herein relate to an assay. Some such embodiments include multiplex affinity probes and an antigen probe. multiplex affinity probes and an antigen probe Some embodiments include contacting a biological sample with the probes, wherein target binding agents such as antibodies in the biological sample compete away the multiplex affinity probes from binding to the antigen probe. Some such embodiments include detecting a decrease in binding of the multiplex affinity probes to the antigen probe, thereby indicating the presence or an amount of the target binding agents in the biological sample.

What is claimed is: 
     
         1 . An assay method, comprising:
 (a) providing a first multiplex affinity probe comprising a first affinity binding agent and a first DNA barcode probe;   (b) providing a second multiplex affinity probe comprising a second affinity binding agent and a second DNA barcode probe;   (c) providing an antigen probe comprising a first epitope recognizable by the first affinity binding agent, a second epitope recognizable by the second affinity binding agent, and a third DNA barcode probe;   (d) contacting a biological sample with the antigen probe, wherein a first target antibody binds to the first epitope to form a first target antibody epitope complex if the biological sample comprises the first target antibody, and wherein a second target antibody binds to the second epitope to form a second target antibody epitope complex if the biological sample comprises the second target antibody;   (e) contacting the biological sample with the first and second multiplex affinity probes, wherein the first multiplex affinity probe binds to the first epitope to form a first multiplex affinity probe epitope complex if the biological sample does not comprise the first target antibody, and wherein the second multiplex affinity probe binds to the second epitope to form a second multiplex affinity probe epitope complex if the biological sample does not comprise the second target antibody; and   (f) detecting the presence or absence of the first and second multiplex affinity probe epitope complexes using a polymerase chain reaction (PCR),   wherein the first and second affinity binding agent comprises an antibody or scFv thereof.   
     
     
         2 . The assay method of  claim 1 , wherein detecting the presence or absence of the first and second multiplex affinity probe epitope complexes comprises detecting the presence or absence of the first and second DNA barcode probes. 
     
     
         3 . The assay method of  claim 1 , wherein the biological sample comprises the first target antibody and/or the second target antibody, wherein the first target antibody binds to the first epitope to form a first target antibody epitope complex and wherein the second target antibody binds to the second epitope to form a second target antibody epitope complex, wherein the first multiplex affinity probe binds to the first epitope to form a first multiplex affinity probe epitope complex and/or wherein the second multiplex affinity probe binds to the second epitope to form a second multiplex affinity probe epitope complex, and wherein detecting the presence or absence of the first and second multiplex affinity probe epitope complexes comprises detecting the presence of the first and/or second multiplex affinity probe epitope complexes. 
     
     
         4 . The assay method of  claim 1 , wherein the antigen probe comprises a recombinant protein comprising the first and second epitopes. 
     
     
         5 . The assay method of  claim 1 , further comprising hybridizing the first and third DNA barcode probes with a first connector nucleic acid, wherein the first connector nucleic acid comprises sequences complementary to at least a portion of the first and third DNA barcode probes. 
     
     
         6 . The assay method of  claim 5 , further comprising ligating the first and third DNA barcode probes together to form a first ligation product. 
     
     
         7 . The assay method of  claim 6 , further comprising amplifying a region of the first ligation product using amplification primers specific for the first and third DNA barcode probes, to produce a first amplification product; and detecting or quantifying the first amplification product. 
     
     
         8 . The assay method of  claim 1 , further comprising hybridizing the second and third DNA barcode probes with a second connector nucleic acid, wherein the second connector nucleic acid comprises sequences complementary to at least a portion of the second and third DNA barcode probes. 
     
     
         9 . The assay method of  claim 8 , further comprising ligating the second and third DNA barcode probes together to form a second ligation product. 
     
     
         10 . The assay method of  claim 9 , further comprising amplifying a region of the second ligation product using amplification primers specific for the second and third DNA barcode probes, to produce a second amplification product; and detecting or quantifying the second amplification product. 
     
     
         11 . The assay method of  claim 1 , further comprising hybridizing the first and third DNA barcode probes with each other, wherein the first and third DNA barcode probes are complementary or each comprise a complementary region with each other, and/or further comprising hybridizing the second and third DNA barcode probes with each other, wherein the second and third DNA barcode probes are complementary or each comprise a complementary region with each other. 
     
     
         12 . The assay method of  claim 11 , further comprising contacting the first and third DNA barcode probes with a DNA polymerase, wherein the DNA polymerase forms a first double-stranded DNA molecule from the first and third DNA barcode probes, and/or further comprising contacting the second and third DNA barcode probes with a DNA polymerase, wherein the DNA polymerase forms a second double-stranded DNA molecule from the second and third DNA barcode probes. 
     
     
         13 . The assay method of  claim 12 , further comprising amplifying the first double stranded DNA molecule to produce a first amplification product, and/or further comprising amplifying the second double stranded DNA molecule to produce a second amplification product; and detecting or quantifying the first amplification product, and/or detecting or quantifying the second amplification product. 
     
     
         14 . The assay method of  claim 1 , wherein the first and second multiplex affinity probes are each bound or conjugated to a solid support and/or the antigen probe is bound or conjugated to a solid support or substrate. 
     
     
         15 . The assay method of  claim 1 , wherein the presence of the first or second target antibodies in the biological sample are indicative of the presence of a disease. 
     
     
         16 . The assay method of  claim 15 , wherein the disease comprises a cancer, a hematological disease, diabetes, an autoimmune disease, a dengue virus infection or Zika virus infection. 
     
     
         17 . The assay method of  claim 1 , wherein the biological sample comprises whole blood, serum, plasma, urine, saliva, breast milk, nasal fluids, or cerebrospinal fluid.